RESUMO
【Objective】 To investigate the effects of platelet activation pathways on the characterization of platelet-derived extracellular vesicles(PEVs). 【Methods】 Whole blood from healthy donors was prepared into platelet suspension by centrifugation. Platelets were randomly divided into six groups, the ctrl group added no extra stimulus, and the other five groups were treated with collagen, adenosine diphosphate(ADP), thrombin, Ca2+ ionophore and freeze-thaw cycles (F-T) to activate platelets. Platelet-derived exosomes(PEXOs) and microvesicles(PMVs) were isolated by differential centrifugations, and then were determined by nano-flow cytometry and electron microscopy. The protein markers of PEXOs also were identified by western-blot. The protein concentration and content of PEXOs were also detected. 【Results】 CD9, CD81, TSG101 proteins were detected in all of the PEXOs, which had no calnexin. Both PEXOs and PMVs had CD41; PEXOs were cup-holder-like bilayer membrane vesicles under a transmission electron microscope, while PMVs were irregular membranous structure; 85%-95% of PEXOs were<100nm, 87%-94% of PMVs were 100-300nm. The concentration of exosomes and microvesicles in the F-T group was the highest(205.67±65.27 and 102.73±15.48), followed by the Ca2+ ionophore group(44.42±17.07 and 11.4±4.81). Although in the same size range, the numbers of PEVs induced by different activation conditions varied. The protein concentration of PEXOs in the F-T group(1.11±0.51) was higher than that in the control group(0.32±0.39), ADP group(0.41±0.31) and thrombin group(0.38±0.37), while the total protein(125.40±58.32) was higher than that in other three groups(the ctrl group 25.53±25.96 vs ADP group 37.21±15.73 vs thrombin group 36.28±24.18 vs Ca2+ ionophore group 47.09±23.29). 【Conclusion】 The biological characteristics of PEVs are affected by platelets activation pathways, whose ability to induce protein-packing into exosomes may also be relevant to the particle size. The freeze-thaw cycles can induce high concentrations of extracellular vesicles, which may be an ideal method for the preparation of PEVs.